1. Field of the Invention
The present invention relates to a photothermal conversion spectroscopic analysis method and photothermal conversion spectroscopic analysis apparatus for analyzing a sample using detecting light that has passed through a thermal lens produced in the sample, and in particular to a photothermal conversion spectroscopic analysis method and photothermal conversion spectroscopic analysis apparatus that enable high-accuracy ultramicroanalysis to be carried out in a very small space.
2. Description of the Related Art
In recent years, spectroscopic analysis has come to be widely used as a method for carrying out analysis or measurement on semiconductors, biological samples, various types of liquid sample, and so on. However, with a conventional spectroscopic analysis method, in the case of analyzing a very small amount of a substance in a very small space, a vacuum environment is often required as a measurement condition, and there has been a problem that to realize such an environment the apparatus must be made large in size and the cost increases, and furthermore there has been a problem that the sample may be damaged or destroyed by an electron beam or ion beam used. Moreover, when handling an extremely small amount of a sample in a solution, biological tissue, or the like, it becomes essential to use an optical microscope, which enables high-accuracy analysis to be carried out with high spatial resolution. The only type of microscope actually used in this optical field is a laser fluorescence microscope, and hence the target of analysis is limited to being a laser fluorescence microscope fluorescent molecule, which is inconvenient.
Consequently, there have been demands for an analysis method according to which the target of analysis is not limited to being a fluorescent molecule, a vacuum environment is not required, analysis can be carried out without contacting or damaging the sample, and high-accuracy analysis can be carried out with high spatial resolution.
A photothermal conversion spectroscopic analysis method that uses a thermal lens effect brought about by photothermal conversion is attracting attention as an analysis method that satisfies these demands.
This photothermal conversion spectroscopic analysis method uses a photothermal conversion effect in which a solute in a solution absorbs convergently irradiated light and thus emits thermal energy, the temperature of the solvent is locally raised by this thermal energy and hence the refractive index of the solvent changes locally, and as a result a thermal lens is formed in the solvent.
FIG. 4 is a view useful in explaining the principle of a thermal lens.
In FIG. 4, exciting light is convergently irradiated onto an extremely small sample via an objective lens, whereby a photothermal conversion effect is brought about. For most substances, the refractive index drops as the temperature rises, and hence in the sample onto which the exciting light has been convergently irradiated, the refractive index drops due to the rise in temperature in the vicinity of the center of the converged light. Due to thermal diffusion, the rise in temperature becomes smaller, and hence the drop in refractive index becomes smaller, with increasing distance from the center of the converged light. Optically, the resulting refractive index distribution produces exactly the same effect as a concave lens, and hence the effect is referred to as the thermal lens effect. The degree of the thermal lens effect, which corresponds to the power of the concave lens, is proportional to the optical absorbance of the sample. Moreover, in the case that the refractive index increases with temperature, a similar effect is produced, but the thermal lens is convex.
In the photothermal conversion spectroscopic analysis method described above, thermal diffusion, i.e. change in refractive index, is measured, and hence the method is suitable for detecting concentrations in extremely small samples.
A photothermal conversion spectroscopic analysis apparatus that carries out the photothermal conversion spectroscopic analysis method described above is disclosed, for example, in Japanese Laid-open Patent Publication (Kokai) No. 10-232210.
In such a photothermal conversion spectroscopic analysis apparatus, the sample is disposed below the objective lens of a microscope, and exciting light of a predetermined wavelength outputted from an exciting light source is introduced into the microscope, whereby the exciting light is convergently irradiated via the objective lens onto an extremely small region in the sample. A thermal lens is thus formed, with the center of the thermal lens being at the center of the converged light.
Moreover, detecting light that is outputted from a detecting light source and has a wavelength different to the exciting light passes through the microscope and is thus convergently irradiated onto the thermal lens, whereby the detecting light passes through the thermal lens in the sample and is thus diverged or converged. The diverged or converged detecting light exiting from the sample is taken as signal light, and is passed through a converging lens and a filter or through just a filter, before being detected by a detector. The intensity of the detected signal light depends on the power of the thermal lens formed in the sample.
The detecting light may have the same wavelength as the exciting light, or the exciting light may also be used as the detecting light. However, in general better sensitivity is obtained in the case that the exciting light and the detecting light have different wavelengths to one another.
However, before now studies had not been carried out into how the wavelengths of the exciting light and the detecting light should be selected to enable high-sensitivity measurement to be carried out.
Moreover, in the conventional photothermal conversion spectroscopic analysis apparatus described above, the optical system for the light sources, the measurement part and the detection part (photoelectric conversion part) is complex in arrangement and/or construction, and hence the apparatus is large in size and portability is poor. There has thus been a problem that when carrying out analysis or a chemical reaction using the photothermal conversion spectroscopic analysis apparatus, there are limitations with regard to the installation site and the operation of the photothermal conversion spectroscopic analysis apparatus.
Moreover, in the conventional photothermal conversion spectroscopic analysis apparatus described above, the exciting light and the detecting light are led to the sample through open space, and hence various optical system components such as the light sources, mirrors and lenses must be fixed onto a sturdy baseplate so that these components do not move during measurement. Furthermore, the optical axes of the exciting light and the detecting light may shift out of alignment upon changes in the installation environment of the photothermal conversion spectroscopic analysis apparatus such as temperature, and hence jigs for adjusting for such shifts are required. These jigs are also a cause of the photothermal conversion spectroscopic analysis apparatus becoming larger in size and the portability worsening.
In most cases of using the photothermal conversion spectroscopic analysis that makes use of a thermal lens, it is necessary for the focal position of the exciting light and the focal position of the detecting light to be different to one another. FIG. 5A is a view useful in explaining the formation position of the thermal lens formed by the exciting light and the focal position of the detecting light in the case that the objective lens has chromatic aberration. FIG. 5B is a view useful in explaining the formation position of the thermal lens formed by the exciting light and the focal position of the detecting light in the case that the objective lens does not have chromatic aberration. In FIGS. 5A and 5B the exciting light and the detecting light have different wavelengths to one another.
In the case that the objective lens 130 has chromatic aberration, as shown in FIG. 5A, the thermal lens 131 is formed at the focal position 132 of the exciting light, and the focal position 133 of the detecting light is shifted by an amount ΔL from the focal position 132 of the exciting light due to the difference in wavelength between the exciting light and the detecting light; the detecting light is thus refracted by the thermal lens 131 and hence changes in the refractive index of the thermal lens 131 can be detected as changes in the focal distance of the detecting light. On the other hand, in the case that the objective lens 130 does not have chromatic aberration, as shown in FIG. 5B, the focal position 133 of the detecting light is almost exactly the same as position of the thermal lens 131, which is formed at the focal position 132 of the exciting light; the detecting light is thus not refracted by the thermal lens 131, and hence changes in the refractive index of the thermal lens 131 cannot be detected.
However, the objective lens of a microscope is generally manufactured so as not to have chromatic aberration, and hence the focal position 133 of the detecting light is almost exactly the same as the focal position 132 of the exciting light, i.e. the position of the center of the thermal lens 131, as described above (FIG. 5B). Changes in the refractive index of the thermal lens 131 thus cannot be detected. There has thus been a problem that every time measurement is carried out, trouble must be taken to either shift the position of formation of the thermal lens from the focal position 133 of the detecting light as shown in FIG. 6A or 6B, or else diverge or converge the detecting light slightly using a lens (not shown) before passing the detecting light through the objective lens so that the focal position 133 of the detecting light is shifted from the thermal lens 131 as shown in FIG. 7.